Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effects of cigarette smoking and exposure to cadmium and lead on phenotypic variability of hepatic CYP2A6 and renal function biomarkers in men

Effects of cigarette smoking and exposure to dietary cadmium (Cd) and lead (Pb) on urinary biomarkers of renal function and phenotypic variability of cytochrome P450 2A6 (CYP2A6) were investigated in a group of 96 healthy Thai men with mean age of 36.7 year (19-57 years). In non-smokers, Cd burden increased with age (r = 0.47, P < 0.001). In current smokers, Cd burden increased with both age (r = 0.45, P = 0.01) and number of cigarettes smoked per day (r = 0.32, P = 0.05). Cd-linked renal tubular dysfunction was seen in both smokers and non-smokers, but Pb-linked glomerular dysfunction was seen in smokers only, possibly due to more recent exposure to high levels of Cd and Pb, as reflected by 30-50% higher serum Cd and Pb levels in smokers than non-smokers (P < 0.05). Exposure to dietary Cd and Pb appeared to be associated with mild tubular dysfunction whereas dietary exposure plus cigarette smoking was associated with tubular plus glomerular dysfunction. Hepatic CYP2A6 activity in non-smokers showed a positive association with Cd burden (adjusted P = 0.38, P = 0.006), but it showed an inverse correlation with Pb (adjusted beta = -0.29, P = 0.003), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. In contrast, CYP2A6 activity in current smokers did not correlate with Cd or Pb, but it showed a positive correlation with serum ferritin levels (r = 0.45, P = 0.01). These finding suggest that Pb concentrations in the liver probably were too low to inhibit hepatic synthesis of heme and CYP2A6 and that the concurrent induction of hepatic CYP2A6 and ferritin was probably due to cigarette smoke constituents other than the Cd and Pb. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Evidence for Concurrent Effects of Exposure to Environmental Cadmium and Lead on Hepatic CYP2A6 Phenotype and Renal Function Biomarkers in Non Smokers

We examined the interrelationships between phenotype of hepatic cytochrome P450 2A6 (CYP2A6), nephropathy, and exposure to cadmium and lead in a group of 118 healthy Thai men and women who had never smoked. Their urinary Cd excretion ranged from 0.05 to 2.36 mug/g creatinine, whereas their urinary Pb excretion ranged from 0.1 to 12 mug/g creatinine. Average age and Cd burden of women and men did not differ. Women, however, on average showed a 46% higher urinary Pb excretion (p < 0.001) and lower zinc status, suggested by lower average serum Zn and urinary Zn excretion compared with those in men. Cd-linked nephropathy was detected in both men and women. However, Pb-linked nephropathy was seen only in women, possibly because of higher Pb burden coupled with lower protective factors, notably of Zn (P < 0.001), in women compared with men. In men, Pb burden showed a negative association with CYP2A6 activity (adjusted beta = -0.29, p = 0.003), whereas Cd burden showed a positive association with CYP2A6 activity (adjusted beta = 0.38, p = 0.001), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. The weaker correlation between Cd burden CYP2A6 activity in women despite similarity in Cd burden between men and women is consistent with opposing effects of Pb and Cd on hepatic CYP2A6 phenotypic expression. A positive correlation between Cd-linked nephropathy (urinary N-acetyl-beta-D-glucosaminidase excretion) and CYP2A6 activity in men (r = 0.39, p = 0.002) and women (r = 0.37, p = 0.001) suggests that Cd induction of hepatic CYP2A6 expression and Cd-linked nephropathy occurred simultaneously.

Effects of chronic exposure to low-level cadmium on renal tubular function and CYP2A6-mediated coumarin metabolism in healthy human subjects

Relationships between cadmium (Cd) body burden, kidney function and coumarin metabolism were investigated using two groups of 197 and 200 healthy Thais with men and women in nearly equal numbers. A mean age of one group was 30.5 years and it was 39.3 years for the other group. Of 397, 20 subjects (5%) excreted urine Cd between 1.4 mug/g and 3.8 mug/g creatinine and these subjects faced 10-15% increase in the probability of having abnormal urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG-uria). The prevalence of NAG-uria varied with Cd body burden in a dose-dependent manner (chi(2) = 22, P < 0.008). Also NAG-nuria was one of the three kidney effect markers tested that showed the greatest strength of correlation with urine Cd in both men and women (r = 0.48 P < 0.001). In addition, urine Cd excretion of men and women showed a positive correlation (r = 0.46 to 0.54. P < 0.001) with urine 7-hydroxycoumarin (7-OHC) excretion which was used as a marker of liver cytochrome P450 2A6 (CYP2A6) enzyme activity. Urinary CA excretion accounted for 25% of the total variation in urine 7-OHC excretion (P < 0.001). These data suggest that Cd may increase the expression of CYP2A6 in liver, resulting in enhanced coumarin metabolism in subjects with high Cd body burden. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Glycine metabolism by plant roots and its occurrence in Australian plant communities

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [N-15]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [N-15]glycine incorporation ranged from 26 to 45% of (NH4+)-N-15 incorporation and was 2- to 3-fold greater than (NO3-)-N-15 incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [N-15]glycine and (NO3-)-N-15 in similar amounts, 18-26% of (NH4+)-N-15 incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of N-15 from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of N-15-label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of N-15 from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.

Directed evolution of mammalian cytochrome P450 enzymes involved in xenobiotic metabolism

Directed evolution of cytochrome P450 enzymes represents an attractive means of generating novel catalysts for specialized applications. Xenobiotic-metabolizing P450s are particularly well suited to this approach due to their inherent wide substrate specificity. In the present study, a novel method for DNA shuffling was developed using an initial restriction enzyme digestion step, followed by elimination of long parental sequences by size-selective filtration. P450 2C forms were subjected to a single round of shuffling then coexpressed with reductase in E. coli. A sample (54 clones) of the resultant library was assessed for sequence diversity, hemo- and apoprotein expression, and activity towards the substrate indole. All mutants showed a different RFLP pattern compared to all parents, suggesting that the library was free from contamination by parental forms. Haemoprotein expression was detectable in 45/54 (83%) of the mutants sampled. Indigo production was less than or comparable to the activities of one or more of the parental P450s, but three mutants showed indirubin production in excess of that seen with any parental form, representing a gain of function. In conclusion, a method is presented for the effective shuffling of P450 sequences to generate diverse libraries of mutant P450s containing a high proportion of correctly folded hemoprotein, and minimal contamination with parental forms.

Haemonchus contortus: The uptake and metabolism of closantel

Closantel is an anthe lmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms. (C) 1997 Australian Society for Parasitology.

Increasing relevance of pharmacogenetics of drug metabolism in clinical practice

Much of the individual variation in drug response is due to genetic drug metabolic polymorphisms. Clinically relevant examples include acetylator status; cytochrome P450 2D6, 2C9 and 2C19 polymorphisms; and thiopurine methyltransferase deficiency. It is important to be aware of which drugs are subject to pharmacogenetic variability. In the future, population-based pharmacogenetic testing will allow more individualized drug treatment and will avoid the current empiricism.

Use of the web to provide learning support for a large metabolism and nutrition class

To support student learning in a large Metabolism and Nutrition class, we have introduced a web-based package, using a commercially available program, WebCT. The package was developed at a minimal cost and with limited resources. In addition to downloadable (PDF) versions of lecture Powerpoint presentations, tutorial outlines and a practical class exercise, web-based self-directed learning exercises were included to reinforce and extend lecture material in an active learning environment. The web-site also contained a variety of formative and summative assessment tasks that examined both factual recall and higher order thinking Detailed course information, timetables and a bulletin board were also readily accessible. Student usage of the site was generally high, but varied widely between individual students. Students who achieved a high overall score for the course completed on average three times as many formative assessment items and achieved a higher score for all tests than students who did poorly. Student feedback about the site was very positive with the majority of students reporting that the course material and assessment items that were available were useful to their learning. Administration of the course was also facilitated. (C) 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

Pectenotoxins - an issue for public health - A review of their comparative toxicology and metabolism

Pectenotoxins (PTXs) are a group of toxins associated with diarrhetic shellfish poisoning (DSP) and isolated from DSP toxin-producing dinoflagellate algae. Consumption of shellfish contaminated with PTXs has been associated with incidences of severe diarrhetic illness resulting in hospitalisation. Concern has been raised for public health following the discovery that these toxins are not only hepatotoxic and can cause diarrhetic effects in mammals, but that they are potently cytotoxic to human cancer cell lines and have been found to be tumour promoters in animals. With advances in knowledge and technology, more PTXs are being identified, but little is known of their toxicology and the potential impact these toxins may have on public health in the long term. Without such information, adequate health-risk assessments for the consumption of shellfish contaminated with PTXs cannot be performed. This review gives a brief introduction to diarrhetic shellfish toxins, details the known toxicology and metabolism of PTXs in animals, and discusses known incidences of PTX poisoning in humans. (C) 2001 Elsevier Science Ltd. All rights reserved.

Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization

Friedreich ataxia (FA) Is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (beta-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and beta-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis. (C) 2002 by The American Society of Hematology.

Intrinsic regional differences in androgen receptors and dihydrotestosterone metabolism in human preadipocytes

Androgens play an important role in regulating the central obesity that is a strong risk factor for cardiovascular disease and insulin resistance. This study confirms that androgen receptors are present in subcultured human preadipocytes, with androgen receptor gene expression and saturable specific dihydrotestosterone binding, dissociation constant 1.02 - 2.56 nM and maximal binding capacity 30.8 - 55.7 fmol/mg protein. There was an intrinsic regional difference in androgen receptor complement, with more androgen receptors in visceral than in subcutaneous preadipocytes. Dihydrotestosterone was metabolised by human preadipocytes, with more androstanediol produced by subcutaneous than visceral preadipocytes. While dihydrotestosterone metabolism was insufficient to explain the regional variation in androgen binding, both of these differences would reduce the androgen responsiveness of the subcutaneous preadipocytes compared with visceral preadipocytes. There were no gender differences in androgen binding or metabolism. While the direct effects of androgens on human PAS remain uncertain, these regional differences suggest that AR-mediated regulation of certain PA functions influences adipose tissue distribution.